Project Summary Synthetic Biology has the potential to positively impact human health is many ways. However, dependency on using chemical synthesis methods has limited the ability to rapidly and accurately generate gene-length products for on-demand production of whole genomes. This is particularly useful for combatting sequence diversity in rapidly mutating viruses for vaccine development to prevent world-wide epidemics. Currently, diminishing yields in oligonucleotide synthesis using the phosphoramidite chemical method limit DNA synthesis lengths to ~200 bases, and incur a very high error rate. Therefore, products of phosphoramidite synthesis must be filtered for deletions using expensive and time-consuming gel purification methods. In addition, to generate an entire gene of >1000 bases, shorter strands must be assembled with polymerase and ligation-based methods to achieve the desired full-length product, which can take many days to accomplish. Thus, there is an unmet need for methods capable of kilobase synthesis to rapidly generate artificial genes for whole genome production. Harnessing the power of an enzyme to produce the DNA is an appealing solution. The only known enzyme fully dedicated to template-independent ssDNA synthesis is terminal deoxynucleotidyl transferase (TdT). In uninterrupted homo- polymerization, TdT can produce > 8000 bases at a rate of 16 bases / minute. However, because its 500 million- year old biological function in the immune system is to increase antigen receptor diversity, TdT only will randomly add bases to the 3? end of DNA. This presents a major challenge in using TdT in controlled, stepwise synthesis of a desired DNA sequence. Therefore, it is necessary to engineer a TdT variant that is ideal for stepwise synthesis using blocked nucleotides as substrates. To accomplish this, a method developed for massively parallel CRISPR-Cas9 editing platform to generate thousands of TdT variants in yeast will be applied, followed by screening of TdT activity with a novel high-throughput reporter system. The reporter system will leverage scalable Illumina sequencing to readout incorporation of bases by TdT at a DNA double strand break. Select TdT variants will then be studied for increased base incorporation efficiencies by measuring steady-state kinetics on purified proteins. In parallel, a DNA synthesizer designed for enzymatic synthesis will be built for synthesizing gene-length strands > 1000 bases using these high-performance TdT variants. By exploiting the power of TdT, our platform has the potential to improve the length, speed, error rate, and cost by orders of magnitude over the traditional phosphoramidite method.